KR100223395B1 - In vitro generation of human dendritic cells and uses thereof - Google Patents

Abstract

The present invention provides a method of producing human dendritic cells in vitro by treating CD34 ⁺ cells with tumor necrosis factor-α and interleukin-3 or GM-CSF. The invention also includes a cellular composition of dendritic cells prepared by the method. Dendritic cells of the present invention have many diagnostic and therapeutic systems, including improved mixed lymphocyte responses for tissue rejection assays, passive immunotherapy of cancer, passive immunotherapy of HIV and other viral infections, and SCID-hu mice for human antibody production. It can be widely used as a component in.

Description

[Name of invention]

In vitro production of human dendritic cells and uses thereof

[Technical Field]

The present invention generally relates to methods for producing human dendritic cells in vitro, and more particularly for therapeutic and diagnostic uses of the cells produced thereby.

Dendritic cells are antigen-presenting cell systems that act to initiate several immune responses, such as sensitization of MHC-suppressed T cells, rejection of organ grafts, and formation of T cell-dependent antibodies. Dendritic cells are found in many non-lymphatic tissues, but can migrate into the T cell-dependent region of lymphatic organs through afferent lymph or blood flow. When they are found in the skin they are called Langerhans cells and are also present in the mucous membranes. These are guard groups of the immune system in peripheral tissues (the tissues from which they can obtain antigens). Since these cells express CD4 and can be infected in vitro by HIV, they seem to provide an inlet for HIV virus in vivo. Knight et al. 145 in Racz. et. al .. editors. Accessory Cells in HIV and Other Retroviral Infections (Karger, Basel, 1991): Ramsauer et al. 155 in Racz. et al .. editors (see above)]. Isolation of human dendritic cells from peripheral blood has only recently been successful, and only a small number of cells can be produced (see Freudenthal et al. Proc. Natl. Acad. Sci. Vol. 87. pp. 7698 (1990). )). The production of large numbers of human dendritic cells in vitro primes human pure CD4 and CD8 T cells in vitro, screens for agents that can interfere with HIV infection, and reconstitutes human T and B cells. It generates important primary and secondary in vivo responses of human B cells in SCID-hu mice and constitutes a more sensitive mixed-lymphocyte response assay.

A major obstacle to the transplantation of allogeneic tissues and organs is the rejection of tissue transplantation by recipients of transplant tissue. The cell-mediated immune response of the recipient or host to the donor tissue plays an important role in the rejection process. Cell-mediated immune responses have two important aspects: (1) cognitive modalities when the host cell recognizes donor cells as foreign in the context of a major histocompatibility complex (MHC); And (ii) a mode of destruction when the host cell attacks and reacts with foreign cells. As part of the attack process, many reactive cells multiply and acquire the cytotoxicity, ie the ability to kill donor cells exhibiting the appropriate antigen. Thus, cell-mediated immunity can be described in terms of two measurable actions: proliferation and cytotoxicity. Dubey et al., Chapter 131 in Rose et al., Editors. Manual of Clinical Laboratory Immunology. 3rd edition (American Society of Microbiology, Washington, D.C., 1986)].

The development of cell culture techniques has led to the establishment of in vitro methods that mimic the in vivo immune process, thereby providing a measure for assessing cell-mediated immunity in vitro. In particular, usefulness in connection with transplantation is mixed lymphocyte response (MLR), or mixed lymphocyte culture. MLR is a relatively simple assay, but there are many variations. Typically, this assay consists of mixing the reaction lymphocytes in a suitable culture system with the stimulating lymphocytes proliferated and / or transcriptional devices neutralized, for example, by irradiation. After culturing the cells for several days, a number of different measurements are made to quantify the responsiveness of reactive cells to stimulating cells, for example, the absorption of tritiated thymidine, the number of blast cells, the number of dividing cells, cytokine production, and the like. Can be. Other variables in this assay include sources of reactive and stimulating cells, such as peripheral blood, spleen, ramp nodes; Whether the reactive cells are syngenic, allogenic or xenogenic to the stimulating cells; Methods of neutralizing stimulating cells, such as irradiation or treatment with DNA synthesis inhibitors such as mitomycin C, and the like.

The drawback of MLR as a common assay for cell-mediated immune reactivity is sensitization. Occasionally, even with a particular reading method, it is difficult to obtain a powerful effect on the MLR. Antigen-presenting cells in a stimulus population are believed to be involved in stimulating response cells: however, in most tissues, a source such as cells is a small and / or inefficient type of stimulation. Sensitization of MLR assays, and thus usefulness, can be significantly improved by using more powerful stimulating cell populations. Since dendritic cells are well known as strong antigen-presenting cells, the cells can be used for this action. Steinman, Ann. Rev. Immunol .. Vol. 9. pgs. 271-296 (1991). Unfortunately, it is currently very difficult to obtain dendritic cells in sufficient amounts for use with conventional MLR. See Steinman. et al., chapter 49 in Herzenberg et al., Editors. Cellular Immunology Vol. 2 (Blackwell Scientific Publications. Oxford. 1986)].

[Summary of invention]

The present invention relates to a method for in vitro production of human dendritic cells. The invention also encompasses the use of isolated cells, including an isolated population of human dendritic cells produced by the methods of the invention, and an improved MLR assay using a pure population of dendritic cells as stimulating cells. The method of the present invention provides CD34 ⁺ hematopoietic progenitor in the presence of tumor necrosis factor-α (TNF-α) and interleukin-3 (IL-3) or in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF). Culturing the cells to form the CD1a ⁺ dendritic cells of the invention.

Accordingly, the present invention provides a method of treating CD34 ⁺ hematopoietic cells with TNF-α and IL-3 or GM-CSF; And

It provides a method for producing a cellular composition comprising human dendritic cells, comprising the step of isolating the CD34 ⁺ hematopoietic cells treated as described above, expressing the CD1a antigen.

Preferably, CD34 ⁺ cells are treated with TNF-α as well as GM-CSF.

The invention also provides a cellular composition comprising CD34 ⁺ hematopoietic cells expressing the CD1a antigen by in vitro culture, and human dendritic cells produced by the method as defined above.

Dendritic cells initiate an immunological response. In vitro data reported herein for dendritic cells according to the present invention indicate that these cells are useful as laboratory tools and have utility in vivo treatment by proton (passive) immunotherapy of various diseases including cancer and viral infections. Indicates that it can.

The invention also relates to the use of TNF-α and IL-3 or GM-CSF to prepare dendritic cell lines or to induce dendritic cells; The use of dendritic cells to produce CD34 ⁺ helper T cells; The use of CD34 ⁺ helper T cells in passive immunotherapy; Use of SCID-hu mice for the production of human antibodies: and the use of dendritic cells to produce cancer-specific and virus-specific CD8 ⁺ cytotoxic T cells, in particular CD8 ⁺ cytotoxic T cells specific for AIDS virus It relates to the use of dendritic cells to generate cells.

Another aspect of the invention provides a method for treating a sample of reactive cells; The stimulating cells are homologous to the reactive cells, wherein the stimulating cells are (a) treating CD34 ⁺ hematopoietic cells with TNF-α and IL-3 or GM-CSF and (b) expressing the CD1a antigen. Providing a sample of stimulating cells inactivated to consist of dendritic cells produced by a method comprising isolating the treated CD34 ⁺ hematopoietic cells;

Culturing the reaction cells and the inactivated stimulation cells together; And

Mixed lymphocyte reaction comprising the step of measuring the reactivity of the reactive cells.

[Brief Description of Drawings]

1 is flow cytometry data showing partial co-expression of CD14 by CD1a ⁺ cells.

2 shows data on the growth kinetics of CD1a ⁺ cells under various media conditions.

3 shows data on CD4 ⁺ cell proliferation after stimulation by dendritic cells of the present invention.

4 shows the effect of CD1a ⁺ cells on stimulation of allogeneic CD4 ⁺ T cell proliferation.

5 shows data on CD4 ⁺ and CD8 ⁺ T cell proliferation after stimulation by dendritic cells of the present invention.

Detailed description of the invention

An important aspect of the present invention is the production of dendritic cells from CD34 ⁺ hematopoietic cells.

CD34 ⁺ hematopoietic cells are obtained from various tissue sources, for example bone marrow, but are preferably obtained from umbilical cord blood samples as follows: Precision monocytes are separated from the sample by Ficoll-Hypaque gradient (d = 1.077). g / ml) and incubated overnight at 37 ° C. in RPMI 1640 medium supplemented with bovine serum albumin, for example, of 1% w / v tissue-culture grade to remove adherent cells. Preferably, cells comprising the CD34 antigen are prepared by anti-CD34 monoclonal antibodies, for example anti-My 10 available from Imu-133.3, Becton Dickinson (Mountain View, California) available from Immunotech (Marseille, France). Isolation from non-adhesive monocyte fractions via positive selection by indirect immune panning using and the like. Panning flasks were prepared as follows: 25 μg / ml Sheep Fab anti-mouse IgG in Tris buffer (0.05 mol / l, pH 9.4) was dispensed into a 75-cm 2 tissue culture flask (10 ml) at 4 ° C. Coat overnight. Separately, 5 μg / ml at 10 ⁷ cells / ml in RPMI 1640 supplemented with pooled human AB serum (HABS) supplemented with 2% heat-inactivated light-density monocytes (which remove adherent cells as described above) Incubate with anti-CD34 antibody at 4 ° C. for 1 hour. The cells are then washed in cold medium containing 2% HABS and 10 ml containing about 5 × 10 ⁷ cells are dispensed into flasks previously coated with positive antimouse IgG as described above. After 2 hours of incubation at 4 ° C., non-adherent cells (ie, CD34-depleted fractions) in suspension are harvested by gently pipetting and washing several times with medium. Subsequently, the panned cells (ie, CD34-rich fractions) are recovered by vigorous pipetting.

Dendritic cells are obtained from CD34 ⁺ cells by culturing in a medium comprising GM-SCF or TNF-α and IL-3. Preferably, dendritic cells are obtained from CD34 ⁺ cells by culturing in a medium comprising GM-CSF as well as TNF-α. TNF-α, GM-CSF and IL-3 suitable for use in the present invention are commercially available, for example, from Genzyme Corp. (Cambridge, Mass.) Or are described, for example, in US Pat. No. 4,959,455 (IL-3); PCT Patent Application EP 85/00326 (International Publication No. W086 / 00639) (GM-CSF) by Clark et al. And US Pat. Nos. 4,677, 063 (TNF-α) to Mark et al. Likewise, it can be prepared by a recombinant expression system. Preferably, IL-3 and GM-CSF are used at saturation concentrations: that is, the concentration at which all IL-3 and GM-CSF receptors on CD34 ⁺ cells are occupied by biologically active IL-3 and GM-CSF molecules Used as Of course, the actual concentration may vary depending on the nature of the IL-3 and GM-CSF used. Preferably, human IL-3 having a specific activity of at least 5 × 10 ⁶ U / mg is used, wherein the active unit is at half maximum as measured by ³ H-thymidine uptake by human bone marrow cells in liquid culture. Corresponds to the proliferative activity. In the culture system described below, the saturation concentration is 10 ng / ml (or 50 U / ml). Preferably, human GM-CSF having a specific activity of at least 2 × 10 ⁶ U / mg is used, wherein the active unit is as defined for IL-3 above. In the culture system described below, the saturation concentration is 100 ng / ml (or 200 U / ml). Preferably, TNF-α is 2 to 3 ng / ml or 40 to 60 U / ml. Most preferably at a concentration in the range of about 2.5 ng / ml or 50 U / ml. The units of TNF-α are described in Carswell et al., Proc. Natl. Acad. Sci., Vol. 72. pg. 3666 (1975). and by Aggarwal et al., J. Biol. Chem .. Vol. 260.pg. 2345 (1985). Cells are standard tissue culture medium containing standard additives, eg 10% (v / v) heat inactivated fetal bovine serum, 10 mM Hepes. It can be co-cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 5 × 10 ⁻⁵ M 2-mercaptoethanol, penicillin (100 U / ml) and streptomycin (100 mg / ml). Preferably, CD34 ⁺ cells are cultured in the presence of cytokines for 8-12 days.

Dendritic cells formed in culture are separated by panning as described above, except that anti-CD1a and / or anti-CD14 antibodies are used (both antibodies are commercially available, for example from Becton-Dickinson). Do).

The MLR method of the present invention comprises the following steps: (1) providing a sample of reactive cells; (2) the stimulating cells are homologous to the reactive cells, and the stimulating cells are treated with (a) treating CD34 ⁺ hematopoietic cells with TNF-α and IL-3 or GM-CSF and (b) expressing the CD1a antigen. Providing a sample of stimulating cells inactivated to consist of dendritic cells produced by the method comprising the step of isolating CD34 ⁺ hematopoietic cells treated as above; (3) co-culturing the reaction cells with the stimulation cells inactivated as above; And (4) measuring the reactivity of the reactive cells.

Preferably, the sample of reactive cells consists of CD4 ⁺ T cells from peripheral blood of the patient who will receive the graft. Techniques used to obtain T cell populations are well known in the art and described in DiSabato et al. Eds .. in Meth. in Enzymol .. Vol. 108 (1984). For example, CD4 ⁺ T cells can be isolated as follows: first monocytes are isolated from peripheral blood and adherent cells are removed. CD4 ⁺ T cells can then be obtained, for example, from commercially available monoclonal antibodies such as anti-CD14, anti-CD16, anti-CD20, anti-CD8, anti-CD40 (Becton-Dickinson and / or Ortho Diagnostic Systems, New Jersey commercial cocktails are used to remove and purify other cell types by immunomagnetisation (eg using Dynabeads (Dynal. Oslo. Norway)). CD4 ⁺ populations with purity greater than 95% are typically obtained after two immunomagnetic ablations.

Stimulator cells of the invention are dendritic cells derived from CD34 ⁺ hematopoietic cells taken from a person other than the person taking the reaction cell. That is, stimulatory cells are homologous to the response cells. These cells are obtained as described above.

Stimulator cells of the present invention are inactivated so that they still contain their stimulatory action but inhibit other actions that may mask the response measured from the response cell. Thus, the nature of the inactivation depends somewhat on the read-out of the assay. Preferably, the response, or reading, measured in the response cell is cell proliferation. Other readings also include phenomena such as cytokine production, cell resolution, and the like. Preferably, the stimulating cells are treated so that they cannot replicate but their antigen-processing machinery is still functional. This is typically done by irradiating the cells with, for example, about 1,500 to 5,000 R (gamma or X-radiation), preferably 3,000 to 4,000 R, prior to mixing with the reaction cells.

Preferably, proliferation of the response cells is measured by uptake of tritiated thymidine according to standard protocols. For example, 10-2.5 × 10 ⁴ stimulation cells are added to 2.4 × 10 ⁴ homologous CD4 ⁺ T cells in 96-well round bottom tissue culture plates and incubated for 4 days in the medium described above. After incubation, the cells were pulsed with 1 μCi of tritiated thymidine for 6 hours and then collected and measured for absorption of tritiated thymidine, for example by scintillation counting.

[Experiment]

The following examples are intended to illustrate the present invention. Cell lines, reagents and their concentrations, temperatures, and other variables are only intended to illustrate the application of the present invention, which is not intended to limit the present invention.

EXAMPLE

Example 1

Generation of human dendritic cells

After incubation for 12 days in GM-CSF and TNF-α, cells generated from CD34 ⁺ cord hematopoietic progenitor cells are processed for 2-color fluorescence measurements. Briefly stated, cells were subsequently serially unconjugated monoclonal antibodies, phycoerythrin-conjugated (PE-conjugated) anti-mouse immunoglobulins, normal mouse serum, and monoclonal antibody OKT6 (anti-CD1a). Ortho) or Leu-M3 (anti-CD14; Becton-Dickinson) (wherein monoclonal antibodies are directly labeled with fluorescein isothiocyanate (FITC)). As shown in FIG. 1, CD34 ⁺ cells were cultured for 12 days in the presence of GM-CSF and TNF-α to emerge CD14 ⁺ monocytes that co-express CD1a antigen. In addition, a consistent population of CD14 ⁻ CD1a ⁺ cells with a total CD1a population of 30-90% (range from five experiments) is consistently observed. In monocytic lineage, the expression of CD1a antigen is limited to Langerhans cells. Figure 1a shows the two-color fluorescence intensity from IgG ₁ -FITC isotype control vs. IgG _2a -PE isotype control. 1B shows the two-color fluorescence intensities from OKT6-FITC (proportional to CD1a expression) vs. Leu-M3-PE (proportional to CD14 expression). As shown in FIG. 2, CD1a ⁺ cells were not detected at the start of culture, and CD1a antigen was observed for the first time after 4 days of culture in the presence of TNF-α, and expression was increased by 20 days. Less than 5% of CD1a ⁺ cells were observed in the presence of IL-3, while 5-15% of CD1a ⁺ cells were detected in the presence of GM-CSF. A large proportion of CD1a ⁺ cells were detected in IL-3 and TNF-α (10-25%) and mainly GM-CSF and TNF-α (20-60%) (range from 5 trials after 12 days of culture). . In terms of growth efficiency, IL-3 + TNF-α was two to three times stronger than IL-3 alone, and GM-CSF + TNF-α was three to four times stronger than GM-CSF alone (FIG. 2). Thus, starting from 10 ⁵ CD34 ⁺ cells, 12 days after culture, 1 to 3 × 10 ⁶ CD1a ⁺ cells were produced in GM-CSF, whereas 2 to 3 × in IL-3 + TNF-α and GM-CSF + TNF-α. 10 ⁶ cells were recovered. Since GM-CSF + TNF-α appears to be the most potent combinatorial factor for CD1a ⁺ cell production, all cultures for the characterization of these cells include GM-CSF + TNF-α: (III) IL-3 (Fig. ), (Ii) augmentation of CD34 ⁺ cells 10 ^{5 in} the presence of IL-3 and TNF-α (Δ), (iii) GM-CSF (■), and (iii) GM-CSF and TNF-α (□). Seems. -Is the total number of cells in three experiments, and---represents the expression of CD1a antigen. 2A and 2B show data from two separate sets of experiments.

The morphology of CD1a ⁺ cells produced by the method of the present invention is examined by light microscopy and electron microscopy. Adhesion cells observed in GM-CSF alone show a general pattern for certain macrophages, whereas cells obtained in the presence of GM-CSF + TNF-α show a chorionic dendritic surface, lobular nucleus, and dendritic protrusions. Have typical aspects of dendritic cells. Some of the CD1a ⁺ cells (about 1 in 5) have organelles that connect the double membranes, reminiscent of the structure of Birbeck granules.

The phenotype of CD1a ⁺ cells produced 12 days after culture in the presence of GM-CSF and TNF-α is determined by two-color fluorescence analysis. Depending on the experiment, the percentage of CD1a ⁺ cells co-expressing CD14 varied from 10 to 70%. The results shown in Table 1 below were obtained from experiments in which less than 15% of CD1a ⁺ cells co-expressed CD14: Thus, both CD1a ⁺ cells and CD1a ⁻ CD14 ⁺ cells were characterized. CD1a ⁺ cells co-expressed CD1c, CD4, and CD40 but did not express CD1b. In contrast, CD1a ^- CD14 ⁺ cells did not express CD1c but only slightly expressed CD4 and CD40. Both CD1a ⁺ and CD14 ⁺ cells contain Fcr RII (CD32), Fcr RIII (CD16), and CR3 (CD11b), whereas only CD1a ⁻ CD14 ⁺ cells express Fcr RI (CD64) and CR1 (CD35) Turned out to be. CD1a ⁺ and CD1a ^- CD14 ⁺ cells expressed both LFA1α (CD11a) and LFA1β (CD18), but CD1a ⁺ cells expressed higher levels of ICAM1 (CD54) than CD1a ⁺ CD14 ⁺ cells. CD1a ⁺ cells expressed very high levels of HLA-DR (5-10 fold more than CD14 ⁺ cells). In contrast, CD1a ^- CD14 ⁺ cells expressed high levels of HLA-DQ ⁺ .

Table 1

Phenotype of Dendritic Cells of the Invention

Antibody sources are as follows: Ort = Ortho Diagnostic Systems; Imu = Immunotech: Cou = Coulter (Hialeah. FL); BD = Becton-Dickinson; Med = Medarex Inc. (Lebanon, NH); Hy 2 = Hybridoma, Vol. 2. pg. 423 (1983); Hy8 = Hybridoma, Vol. 8. pg. 199 (1989); EP = Eur. Pat. Appl. 89 403 491.7

Example 2

Proliferation of CD4 + T Cells After Stimulation by Dendritic Cells

CD34 ⁺ cells were incubated for 12 hours according to the present invention and then irradiated with 4,000 Rad to produce experimental stimulatory cells. 2.5 × 10 ⁴ resting CD4 ⁺ T cells or other reactive cells in round bottom micro test tissue culture plates in medium supplemented with 10% (2.4 × 10 ⁴ ) stimulating cells with 10% human AB ⁺ serum as described below Seeding for.

Dendritic cells of the invention are assayed for their ability to induce proliferation of resting allogeneic CD4 ⁺ T cells (as reactive cells). As shown in FIG. 3a, cells (▲) cultured in the presence of IL-3 alone induced the lowest allogeneic CD4 ⁺ T cell proliferation. In contrast, cells cultured in the presence of GM-CSF alone (■), IL-3 + TNF-α (Δ), and GM-CSF + TNF-α (□) strongly induced the proliferation of allogeneic CD4 ⁺ T cells. Depending on the culture conditions of CD34 ⁺ progenitor cells. Optimal proliferation of CD4 ⁺ T cells was observed for values of different ratios ((stimulatory cells) / (homogenous CD34 ⁺ T cells)]. Compared to the control (CD34 ⁺ T cells without stimulating cells), a 50-fold improvement in tritiated thymidine uptake by CD4 ⁺ T cells was shown by GM-CSF alone, IL-3 + TNF-α, and GM-CSF + TNF, respectively. 1: 3.8 (range 1: 3 to 1:25), 1: 12.5 (range 1:10 to 1:35), and 1: 360 (range 1: 100 to 1 :) for cells cultured in the presence of -α. 400) (range from five experiments).

In the experiment shown in FIG. 3b, CD34 ⁺ cells were cultured in the presence of GM-CSF and TNF-α for all experiments, and the response cells were adult peripheral blood (□), cord blood (△ and ▲), and syngeneic cords. Blood CD4 ⁺ T cells (■). 3b the turn, indicates that the code has been stimulated with an allogeneic CD4 ⁺ T cells are derived from a same magnetic pole blood or adult peripheral blood, approximately Winter CD4 ⁺ T cells is very low (20-fold weaker than allogeneic reaction cells).

After 12 days of culture in the presence of GM-CSF and TNF-α, CD1a ⁺ cells were removed by immunomagnetic ablation (FIG. 4). A strong loss of induction was observed. 50-fold improvement in CD4 ⁺ T cell proliferation was achieved by 1: 200 (three experiment ranges from 1: 200 to 1: 400) and 1: 8 (1: 8 to 1:40 before and after removal of CD1a ⁺ cells, respectively). The ratio of (experimental cell) / (homogenous CD4 ⁺ T cell)] was observed. After irradiation, the following were used as stimulating cells: total population (■), population not included by removing CD1a ⁺ cells (□), and control population (Δ) removed with anti-IgG ₁ isotype antibody.

Example 3

Generation of cancer-specific CD8 cytotoxic T cells for passive immunotherapy

CD34 ⁺ cells are isolated from the peripheral blood of cancer patients as described above and grown in the presence of GM-CSF and TNF-α. Peripheral blood CD8 cells are cryopreserved. After generating dendritic cells, CD8 T cells are thawed and then mixed with cancer cells of the patient. After sensitization, CD8 T cells are expanded in the presence of IL-2, as described, for example, in US Pat. No. 4,690,915 to Rosenberg. Total cells are reinjected into the patient if they exhibit tumor-specific cytotoxic activity. Tumor cells are replaced with specific tumor antigens. Van der Bruggen et al. Science. Vol. 254, pp. 1643 (1991).

Dendritic cells produced in the presence of GM-CSF and TNF-α are potent stimulators of resting allogeneic CD8 ⁺ T cell proliferation.

Dendritic cells generated from CD34 ⁺ cord blood cells are irradiated (4,000 Rad) for 12 days and then used as stimulating cells for resting CD4 ⁺ and CD8 ⁺ T cells. For 2 × 10 ⁴ resting T cells in round bottom microtest tissue culture plates in medium (with or without 20 U / ml IL-2) supplemented with 10% 2.5 × 10 ⁵ stimulation cells with 10% human AB ⁺ serum Seeding. After incubation for 5 days, cells are pulsed with ³ H-thymidine 1 μCi for 8 hours, harvested and counted. The test is run in triplicate and the results are expressed in average number / minute.

The addition of IL-2 to the medium in which CD4 ⁺ and CD8 ⁺ T cells were cultured actually stimulated the proliferation of CD4 ⁺ and CD8 ⁺ T cells, as indicated by ³ H-thymidine uptake. As shown in FIG. 5, CD8 ⁺ cells cultured in the presence of medium alone (□) showed very little proliferation, whereas CD8 ⁺ cells cultured in the presence of IL-2 (■) showed greater proliferation; Moreover, CD4 ⁺ cells cultured in the presence of medium alone (○) showed little proliferation, whereas CD4 ⁺ cells cultured in the presence of IL-2 (•) showed significantly greater proliferation.

Example 4

Development of First and Second Antibody Responses in SCID-hu Mice

SCID-hu mice allow a second response to remind the antigen but not to establish a first response (Moller, The SCID-hu Mouse, Vol. 124 (Munksgaard. Copenhagen. 1991); Duchosal et al. Nature. 355. pp. 258 (1992); and Mosier et al .. Curr. Top. Microbiol. Immunol. Vol. 152. pp. 195 (1989)], believed to be due to the lack of reconstitution of dendritic cell pools. Antigen-pulsed dendritic cells can effectively induce antibody responses in vivo (Sornasse et al. J. Exp. Med. Vol. 175. pp. 15 (1992)). -hu mice are reconstituted from dendritic cells generated in vitro from CD34 cells of human cell donors or from another donor cell with a suitable MHC, generating pulses with a suitable antigen prior to injecting the dendritic cells. If the antibodies against B cells, blood and other reconstituted Isolated from the tube and not killed by culturing in a CD40 system in the presence of Epstein-Barr virus as taught, for example, in Banchereau et al. Science. Vol. 251. pp. 70 (1991). It was.

Example 5

AIDS treatment by passive immunotherapy with in vitro generated dendritic cells

Infection of dendritic cells with HIV in vitro has been shown to cause HIV germination from the cell surface after 3-5 days in cultures containing HIV. Patterson. J. Gen. Virol .. Vol. 68. pgs. 1177-1181 (1987); Macatonia et al. Immunology. Vol. 71. pgs. 38-45 (1989); and Knight et al. Immunol. Lett .. Vol. 19. pgs. 177-182 (1988). Evidence for in vivo infection of dendritic cells by HIV results from the description of infection of the skin with Langerhans cells and a decrease in the amount of MHC type 11 molecules in skin cells. Tschachler et al. J. Invest. Dermatol .. Vol. 88. pgs. 233-237 (1987); and Belsito et al. N. Engl. J. Med .. Vol. 310. pgs. 1279-1282 (1984). Moreover, 3 to 21% of blood dendritic cells from HIV patients contain HIV at an infection level two orders of magnitude greater than that observed in other cell types. Macatonia et al. Immunology. Vol. 71. pgs. 38-45 (1990). Infection of dendritic cells by the virus blocks antigenic expression of T cells, thereby inhibiting T cell recruitment / proliferation necessary for a successful immune response.

CD34 ⁺ cells from HIV patients are used to generate dendritic cells according to the present invention. Dendritic cells are then incubated with the selected antigens and reinjected into HIV patients.

Claims (6)

By treatment of the human CD34 ⁺ hematopoietic cells with GM-CSF, TNF-α and IL-3 or GM-CSF and TNF-α inducing the production by human dendritic (dendritic) cells from said CD34 ⁺ hematopoietic cells; And recovering such human dendritic cells, wherein the method comprises preparing a cellular composition comprising human dendritic cells. Dendritic cells pulsed with cancer or viral antigens are generated in vitro and human CD8 ⁺ T cells with sufficient amount of the in vitro generated dendritic cells to sensitize CD8 ⁺ cytotoxic T cells to induce their proliferation. Processing the; And cancer-specific or virus-specific method for generating a human CD8 ⁺ cytotoxic T cell-specific person, including the step of recovering the CD8 ⁺ cytotoxic T cells, and cancer-specific or virus. The method of claim 2, wherein the CD8 ⁺ T cells are sensitized with a cancer antigen to produce cancer specific CD8 ⁺ cytotoxic T cells. The method of claim 2, wherein the CD8 ⁺ T cells are sensitized with a viral antigen to produce virus specific CD8 ⁺ cytotoxic T cells. The method of claim 4, wherein the CD8 ⁺ T cells are sensitized with the viral antigen of AIDS virus and the resulting CD8 ⁺ cytotoxic T cells are specific for HIV virus. Dendritic cells pulsed with antigen are generated in vitro and human CD4 ⁺ T cells with the in vitro generated dendritic cells in an amount sufficient to sensitize antigen-specific CD4 ⁺ helper T cells to induce their proliferation. Processing the; And including the step of recovering the human antigen-specific CD4 ⁺ helper T cells, antigen-specific human how to generate CD4 ⁺ helper T cells.